Joana Pereira

A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint

A molecular switch in a scaffolding protein enables a misfolded, but partially functional, cystic fibrosis protein to evade a quality control checkpoint. Treating cystic fibrosis by subverting quality control Cystic fibrosis is a genetic disorder that affects mostly lung function and is caused by mutations in the transmembrane protein CFTR (cystic fibrosis transmembrane conductance regulator) that regulates cellular fluid secretion and mucus production. Although most mutant versions of the protein retain some function, the cell recognizes the protein as defective and degrades it. The drug VX-809 (or lumacaftor) improves delivery of mutant CFTR to the cell surface, but the cell tags it for removal and degradation. Loureiro et al. found that triggering a conformational change in the scaffolding protein NHERF1 with Rac1 stimulation enables the interaction between NHERF1 and mutant CFTR, thereby preventing the interaction between mutant CFTR and the enzyme that tags it for degradation. Thus, the amount of the partially functional CFTR at the cell surface was higher in patient lung epithelial cells in culture. The findings may enhance the efficacy of VX-809 in cystic fibrosis patients. The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na+/H+ exchange regulatory factor 1) determined whether the PPQC recognized “rescued” F508del-CFTR (the portion that reached the cell surface in VX-809–treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

Targeted delivery of paromomycin in murine infectious diseases through association to nano lipid systems.

UNLABELLED Treatment of intracellular infections such as those caused by Mycobacterium spp. and Leishmania spp. is often hampered by limited access of drugs to infected cells. This is the case of paromomycin (PRM), an antibiotic with broad spectrum in vitro activity against protozoa and mycobacteria. Association of chemotherapeutics to liposomes is a worthy strategy to circumvent poor drug accessibility. Six different PRM liposomal formulations were produced, physicochemically characterized and biologically evaluated in a macrophagic cell line confirming their adequacy for in vivo studies. Biodistribution profiles of PRM liposomes revealed preferential targeting of the antibiotic to the liver, spleen and lungs, relative to free PRM, which translated into an enhanced therapeutic effect in murine models infected with Mycobacterium avium and Leishmania infantum and an absence of toxic effects. Our findings demonstrate the advantages of associating PRM to liposomes indicating their potential as an alternative therapeutic strategy for mycobacterial and parasite infections. FROM THE CLINICAL EDITOR Infections caused by intracellular organisms such as Mycobacterium and Leishmania remain a significant problem worldwide. Although effective drugs are available, their actions are limited by access into the intracellular compartment. In this article, the authors developed different liposomal formulations as drug carriers of paromomycin and investigated their efficacy in a mouse model. The positive should provide another treatment option for these organisms in the near future.

The third dimension: new developments in cell culture models for colorectal research

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffold-free or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

A multi-integrated approach on toxicity effects of engineered TiO2 nanoparticles

The new properties of engineered nanoparticles drive the need for new knowledge on the safety, fate, behavior and biologic effects of these particles on organisms and ecosystems. Titanium dioxide nanoparticles have been used extensively for a wide range of applications, e.g, self-cleaning surface coatings, solar cells, water treatment agents, topical sunscreens. Within this scenario increased environmental exposure can be expected but data on the ecotoxicological evaluation of nanoparticles are still scarce. The main purpose of this work was the evaluation of effects of TiO2 nanoparticles in several organisms, covering different trophic levels, using a battery of aquatic assays. Using fish as a vertebrate model organism tissue histological and ultrastructural observations and the stress enzyme activity were also studied. TiO2 nanoparticles (Aeroxide® P25), two phase composition of anatase (65%) and rutile (35%) with an average particle size value of 27.6±11 nm were used. Results on the EC50 for the tested aquatic organisms showed toxicity for the bacteria, the algae and the crustacean, being the algae the most sensitive tested organism. The aquatic plant Lemna minor showed no effect on growth. The fish Carassius auratus showed no effect on a 21 day survival test, though at a biochemical level the cytosolic Glutathione-S-Transferase total activity, in intestines, showed a general significant decrease (p<0.05) after 14 days of exposure for all tested concentrations. The presence of TiO2 nanoparticles aggregates were observed in the intestine lumen but their internalization by intestine cells could not be confirmed.

Signaling Pathways Driving Aberrant Splicing in Cancer Cells

Aberrant profiles of pre-mRNA splicing are frequently observed in cancer. At the molecular level, an altered profile results from a complex interplay between chromatin modifications, the transcriptional elongation rate of RNA polymerase, and effective binding of the spliceosome to the generated transcripts. Key players in this interplay are regulatory splicing factors (SFs) that bind to gene-specific splice-regulatory sequence elements. Although mutations in genes of some SFs were described, a major driver of aberrant splicing profiles is oncogenic signal transduction pathways. Signaling can affect either the transcriptional expression levels of SFs or the post-translational modification of SF proteins, and both modulate the ratio of nuclear versus cytoplasmic SFs in a given cell. Here, we will review currently known mechanisms by which cancer cell signaling, including the mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 (PI3)-kinase pathway (PI3K) and wingless (Wnt) pathways but also signals from the tumor microenvironment, modulate the activity or subcellular localization of the Ser/Arg rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) families of SFs.

Molecular epidemiology of rotavirus in four provinces of Angola before vaccine introduction

Angola is a sub‐Saharan country in southern Africa highly affected by diarrhoeal disease with limited epidemiological data regarding etiologic agents. This study was performed during 2012–2013, prior to rotavirus vaccine introduction, with the objective to detect and characterize the rotavirus strains circulating in four provinces of the country: Huambo, Luanda, Zaire, and Cabinda. A high rotavirus detection rate (35%, 117/334) was observed. G1 was the most common G‐genotype (83.6%), whereas P[8] (50.9%) followed by P[6] (38.8%) were the most common P‐types. G1P[8] was identified as the predominant combination (50%), followed by the unusual G1P[6] (29.3%). Strains such G2P[4], G8P[6], G9P[6], and G12P[6] were also found in lower frequencies (5.2–1.7%). The P[6] strains did not cluster in the phylogenetic trees according to their geographic origin or even the corresponding G‐genotype, suggesting a limited number of recent introductions and extensive reassortment events. Our results represent the first report on rotavirus genotype profiles in Angola, showing a wide circulation of the unusual genotype G1P[6], and underline the importance of RV surveillance after the vaccine introduction. J. Med. Virol. 88:1511–1520, 2016.

Goal-directed or aimless? EEG differences during the preparation of a reach-and-touch task

The natural control of neuroprostheses is currently a challenge in both rehabilitation engineering and brain-computer interfaces (BCIs) research. One of the recurrent problems is to know exactly when to activate such devices. For the execution of the most common activities of daily living, these devices only need to be active when in the presence of a goal. Therefore, we believe that the distinction between the planning of goal-directed and aimless movements, using non-invasive recordings, can be useful for the implementation of a simple and effective activation method for these devices. We investigated whether those differences are detectable during a reach-and-touch task, using electroencephalography (EEG). Event-related potentials and oscillatory activity changes were studied. Our results show that there are statistically significant differences between both types of movement. Combining this information with movement decoding would allow a natural control strategy for BCIs, exclusively relying on the cognitive processes behind movement preparation and execution.

Gafchromic XR-QA2 film as a complementary dosimeter for hand-monitoring in CTF-guided biopsies

Computed tomography fluoroscopy (CTF) is a useful imaging technique to guide biopsies, particularly lung biopsies, but it also has the potential for very high hand exposures, despite use of quick-check method and needle holders whenever feasible. Therefore, reliable monitoring is crucial to ensure the safe use of CTF. This is a challenge, because ring dosimeters monitor exposure only at the base of one finger, while the fingertips may be exposed to the highly collimated CT beam. In this work we have explored the possibility of using Gafchromic XR-QA2 self-developing film as a complementary dosimeter to quantify hand exposure during CTF-guided biopsies. A glove used in a previous study and designed to contain 11 TLDs was adapted to include Gafchromic strips 7 mm wide, covering the fingers. A total of 22 biopsies were successfully performed wearing this GafTLD glove under sterile gloves, and the IR reported no difficulty or reduction of dexterity while wearing it. Comparison of dose distributions obtained from digitization of the Gafchromic film strips and absolute Hp(0.07) readings from TLDs showed good agreement, despite some positional uncertainty due to relative movement. Per procedure, doses at the base of the ring finger can be as low as 3%-8% of hand dose maximum. Accumulated dose at the base of the ring finger was four times lower than the dose maximum. PACS numbers: 07.57.Kp, 29.40.-n, 85.25.Pb, 87.57.qp.Computed tomography fluoroscopy (CTF) is a useful imaging technique to guide biopsies, particularly lung biopsies, but it also has the potential for very high hand exposures, despite use of quick‐check method and needle holders whenever feasible. Therefore, reliable monitoring is crucial to ensure the safe use of CTF. This is a challenge, because ring dosimeters monitor exposure only at the base of one finger, while the fingertips may be exposed to the highly collimated CT beam. In this work we have explored the possibility of using Gafchromic XR‐QA2 self‐developing film as a complementary dosimeter to quantify hand exposure during CTF‐guided biopsies. A glove used in a previous study and designed to contain 11 TLDs was adapted to include Gafchromic strips 7 mm wide, covering the fingers. A total of 22 biopsies were successfully performed wearing this GafTLD glove under sterile gloves, and the IR reported no difficulty or reduction of dexterity while wearing it. Comparison of dose distributions obtained from digitization of the Gafchromic film strips and absolute Hp(0.07) readings from TLDs showed good agreement, despite some positional uncertainty due to relative movement. Per procedure, doses at the base of the ring finger can be as low as 3%–8% of hand dose maximum. Accumulated dose at the base of the ring finger was four times lower than the dose maximum. PACS numbers: 07.57.Kp, 29.40.‐n, 85.25.Pb, 87.57.qp

Improving Children's Writing Ability

This paper presents IWA, a platform to aid children when learning how to write. The proposed system offers both tutor and child a certain degree of autonomy. IWA provides tutor and child with different interfaces. The features available to the tutor allow the definition and configuration of repetition exercises comprising letters, numerals and freeform gestures. The child interface supports the child in the task of solving those exercises. The system has been evaluated in two sessions with children. From the evaluation results and the feedback provided by a school teacher we conclude this to be a very promising system towards optimizing the repetition process required for perfecting hand-writing.

Lipid-based nanoformulations of trifluralin analogs in the management of Leishmania infantum infections

AIM To improve the potential of trifluralin (TFL) in the management of Leishmania infantum infections through the synthesis of analogs (TFLA) and incorporation in nanoparticulate drug delivery systems (NanoDDS), liposomes and solid lipid nanoparticles, for selective targeting to leishmania infection sites. MATERIAL & METHODS In vitro screening of 18 TFLA was performed by flow cytometry. NanoDDS were loaded with active TFLA and evaluated for antileishmanial efficacy in mice through determination of parasite burden in liver and spleen. RESULTS The in vitro testing revealed the most active and nontoxic TFLAs, which were selected for the in vivo studies based on high incorporation in liposomes and lipid nanoparticles (>90%). Selected TFLA nanoformulations showed superior antileishmanial activity in mice (parasite burden >80%), over free TFLA and Glucantime. CONCLUSION The modification of TFL structure to obtain active TFLA, together with their incorporation in NanoDDS, improved their in vivo performance against L. infantum infection.